Current methods to select somatic cell mutants of cultured mammalian cells rely exlusively on the use of single cells growing in monolayers or in suspension. We intend to study mutation induction using Chinese hamster V-79 cells in 3-dimensional tissue-like growth as "spheroids". Spheroids of different ages contain different proportions of nutrient-depleted, hypoxic and noncycling cells and can be used to determine the influence of these parameters on the ability of an agent to cause mutation to 6-thioguanine or ouabain resistance. Comparison between results obtained using cells grown as monolayers or spheroids will allow an estimate to be made of the influence of drug diffusion, cell-cycle effects, cell density and cell interaction on mutation induction. Several known mutagens will be evaluated with conventional monolayers and spheroids, including ionizing and ultra-violet radiation, nitroheterocycles (Flagyl and Af-2) alkylating agents (MNNG, EMS), polycyclic aromatic hydrocarbons (DMBA, AAF), and metabolic inhibitors such as 5-fluorouracil. Cell subpopulations within spheroids will be separated based on size or fluorescence using a non-toxic DNA-binding stain, Hoescht 33342; the number of induced mutants as well as the number of DNA strand breaks will be determined within subpopulations of spheroids and compared with results obtained using cells grown as monolayers. Internal standards within each experiment (including AF-2 which requires metabolic "activation" for mutation) will allow comparisons between experiments and between groups of drugs. Since the cell interactions and cell packing of spheroids simulates a tissue-like environment, results with this in vitro model will indicate the role of these factors in mutation induction.